Many serious enterococcal infections are no longer treatable. Vancomycin resistance was the last antibiotic to which all enterococci were susceptible. However, vancomycin resistant isolates began to emerge in the late '80s, and now represent approximately 1 in 7 strains obtained from ICUs. The only therapeutic option available for treating these infections is supportive care, essentially as practiced in the pre-antibiotic era. The emergence of enterococcal strain that are completely resistant to all currently approved antibiotic combinations is an issue of great concern in medicine, to the scientific community, and to the general public. Direction in the search for new therapeutic approaches for treating enterococcal infection will come through a clearer understanding of the factors involved in the pathogenesis of enterococcal disease. One of the few enterococcal factors that has been shown to be associated with isolates from sites of infection, associated with severity of disease outcome, and directly shown to contribute to disease severity in several models of infection, is the cytolysin of Enterococcus faecalis. Although an understanding of the molecular nature of the cytolysin is reaching a sophisticated level, essentially nothing is known about the environmental cues that trigger its expression. Common to 1) the in situ production of a toxin associated with severity of infection, 2) the careful expression of an otherwise lethal substance by a producing organism, and 3) the ability of a cytolysin producing organism to transfer the plasmid encoding cytolysin production and resistance to a sensitive recipient, is the regulation of cytolysin expression. The goal of this proposal is therefore to characterize the molecular basis for regulation of the expression of the cytolysin and its auxiliary functions, including those for toxin maturation, secretion, activation, and immunity.